Preparation And Identification Of Monoclonal Antibodies Specific For Porcine PD-1 And Prokaryotic Expression Of Its Ligand PD-L1

Programmed death 1(PD-1), belonging to CD28 family, is inducibly expressed on activated T, B lymphocytes and monocytes. There are two types of its ligands, PD-L1 and PD-L2, respectively. They have great difference in expression patterns. PD-1 can downregulate T cell-mediated proliferation, cytokine induction and cytotoxicity after combining to its ligands, PD-L1 and PD-L2.When PD-1-PD-L pathway is blocked by antibodies against PD-1 or PD-L, the number of effective T cells grows and the effects are also enhanced. The biological functions of porcine PD-1 pervade almost every aspect of immune response in vivo, such as autoimmunity, tumor immunity, transplant immunity, chronic viral infectious immunity, parasite immunity and allergy. Some pathogens can induce persistent infection in pigs, such as porcine reproductive and respiratory syndrome (PRRS), whose important character is inducing persistent infection and immuosuppression. However, there are no reports about whether the PD-1-PD-L pathway functions in the progression of PRRS until now. Here we tried to make monoclonal antibodies specific for porcine PD-1, and provide a basis for investigating the function and immunologic mechanism of porcine immunosuppressive diseases.We constructed prokaryotic expression vector and eukaryotic expressive vector for porcine PD-1, and induced protein expression in Escherichia coli. Then we purified the prokaryotic expressive protein and immunized BALB/c mouse with it. Finally we prepared and identified its MAb. The results are as follows:1. We constructed the prokaryotic expressive vector pGEX-6P-PD-1, induced and expressed it in Escherichia coli using IPTG. SDS-PAGE and Western-blot analysis showed that we got the fusion protein of right size.2. We obtained porcine PD-1 fusion protein with high purity by electroeluting. The fusion protein was used as immunogen to immune BALB/c mouse. Then the 50% PEG4000 was used to fuse the myeloma cell SP2/0 and antigen stimulated spleen B lymphoblast. And we constructed the eukaryotic expressive vector pcDNA3.1-PD-1 and transfected the plasmid into 293T cells by lipofectamine-2000 to develop the indirect immunofluorescent assay (IFA) for testing the cells survived after fusion.Forty eight hours later, the transfected cells were collected and locked on slide, which was used as antigen slide. Finally, using limited dilution, we got a line of positive hybridoma cells, 2B7. Western blot analysis showed that supernatants of 2B7 reacted with GST-PD-1, but not the GST tag of prokaryotic expressive vector, suggesting that antibodies secreted by 2B7 were specific for porcine PD-1. Flow cytometry detection affirmed that 2B7 can recognize membrane type PD-1. The antibody-serecting ability of 2B7 kept stable after freezing and recovering several times.3. We injected 2B7 hybridoma cells into enterocoelia of the mice that were stimulated using liquid paraffin two weeks before, and got ascites of high titers. The IFA titers of 2B7 were 1:1×210 in supernatant and 1∶2×105 in ascites, respectively. The subtype of monoclonal antibodies was identified to be IgG1 by using Ig subtype test kit.4. We cloned porcine PD-L1, and constructed the prokaryotic expressive vector pET-32a-PD-L1, induced and expressed it in E. coli using IPTG. SDS-PAGE and Western-blot analysis showed that we got fusion protein at the right size. At the same time, we selected the optimal conditions for expressing porcine PD-1 fusion protein by induction of IPTG: shaked cultivation at 37℃, and final concentration of IPTG at 1 mmol/L for 4h.In summary, we got a line of hybridoma cells that can stably secrete monoclonal antibodies against porcine PD-1, thus providing foundation for further investigation on physiological function of the PD-1-PD-L pathway in porcine infectious diseases.